Tissue-specific autoregulation of the stat3 gene and its role in interleukin-6-induced survival signals in T cells

Abstract

Signal transducer and activator of transcription 3 (STAT3) mediates signals of various growth factors and cytokines, including interleukin-6 (IL-6). In certain IL-6-responsive cell lines, the stat3 gene is autoregulated by STAT3 through a composite IL-6 response element in its promoter that contains a STAT3-binding element (SBE) and a cyclic AMP-responsive element. To reveal the nature and roles of the stat3 autoregulation in vivo, we generated mice that harbor a mutation in the SBE (stat3(mSBE)). The intact SBE was crucial for IL-6-induced stat3 gene activation in the spleen, especially in the red pulp region, the kidney, and both mature and immature T lymphocytes. The SBE was not required, however, for IL-6-induced stat3 gene activation in hepatocytes. T lymphocytes from the stat3(mSBE/mSBE) mice were more susceptible to apoptosis despite the presence of IL-6 than those from wild-type mice. Consistent with this, IL-6-dependent activation of the Pim-1 and junB genes, direct target genes for STAT3, was attenuated in T lymphocytes of the stat3(mSBE/mSBE) mice. Thus, the tissue-specific autoregulation of the stat3 gene operates in vivo and plays a role in IL-6-induced antiapoptotic signaling in T cells.

FIG. 1

Generation of knockin mice. (A) Schematic diagram of the endogenous locus for the stat3 gene, knockin vectors, and targeted allele. The knockin vectors have a splice acceptor (SA)-β-geo gene flanked with two loxP sequences in the first intron (pBS-stat3wtβ-geo). The mutant promoter has a mutated STAT3-binding site with an NcoI site (underlined, left panel, at bottom). Arrowheads indicate the bases that were altered. The alleles for the targeting vectors were designated stat3^wtSBEβ-geo and stat3^mSBEβ-geo, respectively. The SA and β-geo cassette were removed by crossing stat3^mSBEβ-geo mice with CAG-cre transgenic mice, resulting in the generation of the stat3^mSBE allele (right panel, at bottom). E, EcoRI; N, NcoI; Spe, SpeI; B, BamHI; Sph, SphI; TK, thymidine kinase. (B) Loss of IL-6 responsiveness of the mutated STAT3-binding site. We made reporter constructs containing the minimal junB promoter and the luciferase gene (Luc) with either the wild-type STAT3-binding site from the 5′ region of the stat3 gene, wtSBE Luc, or the mutated binding site, mSBE Luc (left). These were transiently transfected into HepG2 cells and stimulated with IL-6 (open bars) for 5 h or left unstimulated (solid bars). Luciferase activity was normalized to β-galactosidase activity, and averages from triplicate experiments are shown (right). (C) Southern blot analysis of targeted ES clones. BamHI-digested DNA from wild-type ES clones (+/+), targeted clones with the wild-type promoter (stat3^{+/wtSBEβ-geo}), and the mutated promoter (stat3^+/mSBEβ-geo) were hybridized with the 3′ probe shown in A (right panel, at top). (D) Detection of the mutated STAT3-binding site by Southern blot. NcoI-digested DNA from stat3^{+/wtSBEβ-geo} and stat3^+/mSBEβ-geo ES clones was hybridized with the internal probe shown in A (right panel, at top).

FIG. 2

Promoter activities of the stat3 gene in vivo. β-Galactosidase activities were determined by X-Gal staining. Frozen sections were obtained from stat3^{+/wtSBEβ-geo} (left panel) and stat3^+/mSBEβ-geo (right panel) mice. X-Gal staining was performed with sections from the liver (A and B), kidney (C and D), and thymus (E and F) by incubation with a 0.1% X-Gal solution overnight at room temperature.

FIG. 3

Promoter activity of the stat3 gene in the spleen. (A) Expression of β-galactosidase driven by the stat3 promoter in the spleen. IL-6 (30 μg) was injected intravenously into 6-week-old stat3^{+/wtSBEβ-geo} and stat3^+/mSBEβ-geo mice (lower panels), or mice were left untreated (upper panels). Five hours after the injection, the mice were sacrificed and their spleens were removed and sectioned. The expression of β-galactosidase was detected by X-Gal staining for 5 h.(B) Detection of stat3 promoter activity in CD31- and CD11b-positive cells. A section of the spleen from stat3^{+/wtSBEβ-geo} mice was doubly stained with anti-β-galactosidase (green) and antibodies recognizing cell surface markers for CD4, CD45R, CD31, and CD11b (red). The fluorescence images are shown superimposed.

FIG. 4

Generation of stat3^mSBE/mSBE mice. (A and B) Southern blot analysis of the stat3^mSBE allele. The stat3^+/mSBE mice were generated by mating stat3^+/mSBEβ-geo mice with CAG-cre transgenic mice (A), and stat3^mSBE/mSBE mice were generated by intercrossing the stat3^+/mSBE mice (B). Genomic DNA from wild-type and mutant mice was digested with either EcoRI (A and B, upper panel) or NcoI (B, lower panel) and subjected to Southern blot analysis with the internal probe (see Fig. 1A, right panel at top). (C) Induction of stat3 mRNA in stat3^mSBE/mSBE mice. IL-6 was injected intravenously into wild-type and stat3^mSBE/mSBE mice. Three hours after the injection, the mice were sacrificed, and the spleen, kidney, and liver were removed to analyze the levels of stat3 mRNA by Northern blot analysis. The amounts of RNA loaded were evaluated with ethidium bromide staining of 28S and 18S rRNAs.

FIG. 5

Low IL-6 responses of stat3 gene and target genes for STAT3, Pim-1, and junB in thymocytes from stat3^mSBE/mSBE mice. (A) stat3 mRNA expression in thymocytes of stat3^mSBE/mSBE mice. Thymocytes from wild-type and stat3^mSBE/mSBE mice were stimulated in vitro with IL-6 (100 ng/ml) for the indicated periods. Total RNA was examined for stat3 mRNA expression levels by Northern blot analysis. Even loading was evaluated with ethidium bromide staining of 28S and 18S rRNAs. (B) Expression of Pim-1 and junB mRNAs, two target genes for STAT3. Total RNA was prepared from thymocytes that were obtained from wild-type and stat3^mSBE/mSBE mice, without (lanes 0) or with stimulation of IL-6 in vitro for 3 h. The RNA samples were examined for Pim-1 and junB mRNA expression by Northern blot analysis.

FIG. 6

SBE-mediated stat3 autoregulation is involved in IL-6-dependent proliferation and survival of T cells. (A and B) Proliferation of T cells from stat3^mSBE/mSBE mice. Thymocytes (A) and splenic T cells (B) from wild-type (solid bars) and stat3^mSBE/mSBE mice (open bars) were stimulated with anti-CD3 antibody and/or IL-6 for 72 h or left unstimulated (none). Proliferation of these cells was analyzed by labeling them with [³H]thymidine and measuring the incorporated [³H]thymidine with a liquid scintillation counter. (C and D) IL-6-dependent survival of T cells from stat3^mSBE/mSBE mice. Splenic T cells (C) and LN T cells (D) were isolated and cultured with or without IL-6 for 24 h. The cells were stained with PI and FITC-conjugated annexin V and subjected to flow cytometric analysis. The data are divided into quadrants, and the percentage of the entire cell population of the following fractions is indicated in the following quadrants: PI high and annexin V high, PI low and annexin V low, and PI low and annexin V high. There were very few cells that were PI high and annexin V low, and no percentage is given for that quadrant. Representative data from three independent experiments are shown.